Dipeptidyl aminopeptidases (DAP's) are a family of exopeptidases which hydrolize dipeptides from the N-termini of various peptides. The proposed research would utilize DAP I and DAP IV as investigative tools with which to gain information on the primary structure of polypeptides and proteins. Since the specificities of DAP I and DAP IV are nearly complementary, they may be used both in a mixture and separately to provide specific information. The dipeptides released by these enzymes are analyzed by combined gas chromatography/mass spectrometry (GC/MS) after derivatization to form the perfluoropropionyl, methyl ester analogs. It is proposed that the DAP-GC/MS method be more fully developed and, at the same time, its role as a tool for the protein chemist assessed. Work would include purification of the enzymes to eliminate other known protein constituents, investigation of unknown catalytic properties (investigate activity toward glycoproteins, chemically modify residues, uncommon sequences, etc.), insolubilization, and refinements of the GC/MS analytical techniques. Use of the developed methods would include determination of the homology of a series of polypeptides, sequencing by dipeptide overlap, sequence checking of polypeptides sequenced by classical methods, and identification of the position and extent of reaction of modified residues by a dipeptide nearest neighbor analysis. Several specific research projects which require this type of information will be investigated.